ABSTRACT All biological transactions in microbes, plants and mammals share basic processes of DNA replication, DNA transcription, or DNA repair. In diverse organisms, dynamic assemblies of protein carry out the transaction. Although common proteins are sometimes shared, they act as part of unique machines that are specific to the organism or situation. For example, all organisms use the mismatch repair recognition proteins, MSH2-MSH3, to protect the genome by removing extrahelical DNA loops in their DNA. However, specifically at long CAG/CTG DNA tracts, MSH2-MSH3 fails to remove loops which, in humans, cause CAG amplification underlying more than 20 fatal diseases. There is intense interest in determining the mechanisms of the unique machines that result in faulty repair, but missing is a method to resolve them from other sites in the genome where MSH2-MSh3 is working normally. In this proposal, we will develop and apply ?SPOTCHECK? a new, targeted approach to detect site-specific DNA binding partners based on an engineered ascorbate peroxidase (APEX2). SPOTCHECK probes dynamic protein-DNA complexes that form at specific sites during a biological transaction of interest. Briefly, we will insert a TET-operator binding site with CRISPR adjacent to the CAG/CTG repeats, which provides a landing pad for an engineered APEX2-TET-repressor fusion protein. Upon addition of biotin-phenol and hydrogen peroxide, APEX2 covalently tags surrounding endogenous proteins with biotin after formation of the peroxide by biotin-phenoxyl radical oxidation. Since APEX2 is targeted to the CAG/CTG tract, specific tagging of proteins occurs only at the expansion site. The biotin-labeled proteins will be captured on streptavidin magnetic beads, and identified by mass spectrometry (MS). We will use SPOTCHECK to capture the dynamic MSH2-MSH3 complexes ?caught in the act? of expanding the site. The method distinguishes the mutation-causing MSH2- MSH3 complex at the expansion site from a normal MSH2-MSH3 complex that operates simultaneously at other genomic sites.